Several studies have suggested that patients with chronic fatigue syndrome (CFS) have altered immune systems, including peripheral blood mononuclear cell (PBMC) subsets with markers of lymphocyte activation, increased levels of soluble cytokines or markers of inflammation, or alterations in natural killer (NK) cell activation or activity. The first goal of this project is to characterize the frequency of such findings among a unique cohort of patients, monozygotic (MZ) twins discordant for CFS. Using the patient populations described in Projects 1 and 2 we will perform detailed studies measuring phenotypic and functional activity of T, B and NK lymphocytes among patients with CFS and their genotypically identical siblings. If CFS represents an ongoing infection or is related to an autoimmune or chronic T cell disorder initiated by an infectious agent, persistent antigen-specific T lymphocytes may be present in the blood. To address this hypothesis, we will measure the distribution of T lymphocyte beta chain variable region (V beta) gene family utilization within the CD4+ and CD8+ subsets of PBMC from patients with CFS and their twins. Similarly, we will characterize the cytokine responses of disease- discordant and healthy MZ twin pairs. Cytokines will be studied by immunoassays and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD4+ and CD8+ PBMC. Increased antibody titers to herpesviruses have often been found in CFS patients, and are considered additional evidence of immune imbalance. We will determine antibody titers to HHV-6, HHV-7, EBV, and CMV and quantitate viral DNA levels in PBMC, plasma, and saliva to determine the correlation between immune activation, viral replication, and CFS. The availability of MZ twins will allow us to use methods of subtractive hybridization to identify nucleic acid sequences uniquely present in individuals with CFS. This novel patient population approach will allow assessment of the hypothesis that chronic immune activation plays a pathogenic role in some CFS patients.